This application relates to a method, reagents and kit for diagnosis and targeted screening of mutation in p53 protein and mutation in the gene coding for the p53 protein (herein the "p53 gene") Such mutations are collectively referred to herein as "p53 mutations".
The evidence for the tumor suppressor activity of wild-type p53 is now extensive. Since its discovery in 1979 when it was found to be complexed with the SV40 large T antigen in SV40-transformed rodent cells the significance of p53 has slowly come to light. The protein appears to act as a transcription factor, and may be responsible for apoptosis of pre-cancerous cells. (Ziegler et al., "Sunburn and p53 in the onset of skin cancer", Nature 372: 773-776 (1994)) The DNA sequence of the 11 exons of the p53 gene is now known and close to 1000 papers were published on p53 in 1993.
P53 mutations are significant because they are found in an enormous variety of tumors. Among common tumors, about 70% of colorectal cancers, 50% of lung cancers, and up to 40% of breast cancers carry p53 gene mutations. p53 is also linked to cancers of the blood and lymph nodes, including Hodgkin's disease, T cell lymphoma and certain kinds of leukemia. Moreover, aberrant forms of the p53 gene are correlated with more aggressive tumors, metastasis and lower 5-year survival rates. Such reports have emerged for cancers of the colon, lung, cervix, bladder, prostate, breast and skin.
The serious consequences of p53 mutations mandates a method for detection and diagnosis of such mutations which is rapid and accurate and that can be performed at the earliest stage of tumor development. Toward this end, immunoassays and DNA assays for p53 mutations are known in the art. To date, however, neither method has been able to identify p53 mutations with a high degree of specificity and accuracy in a cost effective fashion. Those immunoassays that have been published identify only a small portion of those patients actually thought to be carrying the mutation.
Two general techniques of immunoassay have been employed. The first technique is an indirect method which detects anti-p53 antibodies that arise in some patients who have p53 mutations. Immunoassay tests for detecting anti-p53 antibodies in patient sera are currently based on radioactive labeling, immunoprecipitation and immuno-blotting. All these methods are qualitative and time consuming and thus not suitable for screening large number of samples. (Angelopoulou et al., "Autoantibodies against the p53 tumor suppressor gene product quantified in cancer patient serum with time-resolved immunofluorometry", Cancer J6(6): 315-321 (1993)).
The second technique of immunoassay directly detects mutant p53 protein. Such methods have been disclosed in at least two publications. Bartek et al. disclosed an enzyme-linked immunosorbent assay for p53 which was applied for the measurement of p53 in tumor tissue extracts (Oncogene, 6:1699-1703 (1991)). Hassapoglidou et al. developed a monoclonal antibody useful for direct detection of mutant p53. (Oncogene 8:1501-1509 (1993).)
DNA analysis of p53 mutations has been reported using at least two techniques. First, single stranded conformational polymorphism was used to detect mutant p53 by Kuypers et al. ("Detection of point mutations in DNA using capillary electrophoresis in a polymer network", J. Chromatography 621:149-56 (1993)) and by Felix et al ("Absence of hereditary p53 mutations in 10 familial leukemia pedigrees", J Clin Invest 90:653-8 (1992)). Second, actual genomic DNA sequencing diagnosis was performed on relatively small groups of patients by Toguchida et al. ("Prevalence and spectrum of germline mutations of the p53 gene among patients with sarcoma", N Engl J Med 326:1301-8 (1992)) and by Malkin et al. ("Germline mutations of the p53 tumor-suppressor gene in children and young adults with second malignant neoplasms", N Engl J Med 326:1309-15 (1992)). The cDNA sequence of a larger group of patients (over 400) was reported in a Pharmacia LKB meeting publication (Andell et al. "A new approach in automated DNA sequencing to analyse the p53 gene in a large number of breast cancer patients", Pharmacia LKB meeting literature, 1994) These rapid DNA-based techniques have been used to detect mutations, but because they are so labor intensive, that large-scale screening tests are impractical (Harris et al., "Clinical implications of the p53 tumor-suppressor gene", N Engl J Med 329:1318-1327 (1993)).
Thus, the existing methods of diagnosis have been frustratingly unsatisfactory. Researchers have used either immunoassay or DNA analytical methods to diagnose p53 mutation, even though such tests result in numerous false negatives. A method is required for rapid and cost effective diagnosis of p53 mutation in the millions of individuals who develop potentially life threatening malignancies each year.
It is an object of this invention to provide a method for rapid and cost effective diagnosis of p53 mutations in a sample of patients.
It is a further object of this invention to provide DNA sequencing and amplification primers specific for analysis of the p53 gene from a patient sample.
It is a further object of this invention to provide kits of DNA oligonucleotides for amplification and sequencing of the p53 gene of a patient.
It is a further object of this invention to provide a method of generating a p53 mutation report which is used to provide appropriate genetic counseling to the patient and family upon whom the test is performed.